Novel human estrogen receptor-beta

ABSTRACT

The present invention provides isolated nucleic acids encoding full-length human estrogen receptor-β (hERβ), which comprises 530 amino acids. The invention also provides isolated hERβ polypeptides and hERβ-reactive antibodies, including those that specifically recognize amino acids 1-45 of hERβ. The invention also encompasses methods for identifying hERβ-interactive compounds, including agonists, antagonists, and co-activators.

FIELD OF THE INVENTION

[0001] This invention pertains to DNA encoding a novel human estrogenreceptor-β (hERβ), hERβ polypeptides, and methods for expressing andisolating hERβ. The invention also pertains to methods for using hERβ toidentify coactivators and inhibitors as well as tissue-specificestrogens.

BACKGROUND OF THE INVENTION

[0002] The physiological response to steroid hormones is mediated byspecific interactions of steroids with nuclear receptors, which areligand-activated transcription factors that regulate the expression oftarget genes by binding to specific DNA response elements. Thesereceptors comprise (in an aminoterminal-to-carboxyterminal direction) ahypervariable aminoterminal domain that contributes to thetransactivation function; a highly conserved DNA-binding domainresponsible for receptor dimerization and specific DNA binding; and acarboxyterminal domain involved in ligand-binding, nuclear localization,and ligand-dependent transactivation.

[0003] Recently, cDNA was cloned from rat prostate and was shown to havesignificant homology to a previously isolated rat estrogen receptorcDNA. Kuiper et al., Proc. Natl. Acad. Sci. USA 93:5925, 1996. Thisreceptor was designated ERβ to distinguish it from a previously clonedreceptor, ERα. Rat ERβ was shown to be expressed in the prostate,testes, ovary, and thymus, in contrast to ERα, which is most highlyexpressed in the uterus, breast, liver, and pituitary.

[0004] A human ERβ homologue having the aminoterminal sequenceGly-Tyr-Ser has been reported. Mosselman et al., FEBS Letts. 392:49,1996. This reported sequence lacks an initiator methionine, however;therefore, the complete aminoterminal sequence could not be determined.Thus, the full-length human gene remained unknown and an accuratepicture of the molecular determinants of the transactivation function ofauthentic hERβ could not be obtained.

SUMMARY OF THE INVENTION

[0005] The present invention provides nucleic acids encoding afull-length human estrogen receptor-β (hERβ_(L)). The nucleic acidsequence of hERβ_(L), which is depicted in FIG. 3, SEQ ID NO:1, encodesa receptor having the amino acid sequence depicted in FIG. 4, SEQ IDNO:2. hERβ_(L) according to the present invention contains 45 aminoacids at its aminoterminus which were not previously known. These aminoacids are believed to contribute to the transcription activationfunction of the receptor.

[0006] hERβ_(L) is selectively expressed in the thymus, spleen, ovary,and testes. Accordingly, hERβ can be used to identify co-activatorproteins that are involved in estrogen-regulated gene expression, aswell to identify tissue-selective estrogens.

[0007] The present invention provides isolated polypeptides having thesequence of SEQ ID NO:2 and function-conservative variants thereof whichexhibit estrogen-regulated transcriptional activation activity. In arelated aspect, the invention encompasses isolated peptides derived fromhERβ comprising a sequence corresponding to amino acids 1-45 of SEQ IDNO:2 and function-conservative variants thereof, as explained above. Itis believed that this sequence provides at least part of thetransactivation function.

[0008] The present invention also provides isolated nucleic acidsencoding hERβ_(L) and hERβ_(L)-derived peptides, including the nucleicacid sequence depicted in FIG. 3, SEQ ID NO:1 and subfragments thereofencoding peptides which comprise amino acids 1-45, as well assequence-conservative and function-conservative variants thereof. Alsoencompassed by the invention are DNA vectors comprising anhERβ_(L)-encoding sequence operably linked to a transcription regulatoryelement and cells comprising these vectors. Methods for producinghERβ_(L)-derived polypeptides include incubating a cell comprising anhERβ_(L)-encoding expression vector under conditions that permitexpression of one or more hERβ polypeptides. The methods furtherinclude: (a) harvesting the cells to produce a cell fraction and amedium fraction; and (b) recovering the polypeptide(s) from the cellfraction, medium fraction, or both.

[0009] In another aspect, the invention provides methods for identifyinghERβ-interactive compounds, including agonists, antagonists, andco-activator proteins. In one embodiment, the method includes:

[0010] (a) contacting purified hERβ with a labeled ligand in thepresence of test compounds, to form test reactions, and in the absenceof test compounds, to form control reactions;

[0011] (b) incubating the test and control reactions under appropriateconditions to achieve specific binding of the labelled ligand to hERβ;

[0012] (c) determining the level of binding of the labeled ligand tohERβ in said test and control cultures; and

[0013] (d) identifying as a hERβ-interactive compound any compound thatreduces the binding of the labeled ligand to hERβ.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a schematic illustration of the oligonucleotides usedfor PCR amplification of human estrogen receptor-β (hERβ_(L)) cDNA.

[0015]FIG. 2 is a schematic illustration of the pcDNA3 plasmidcontaining hERβ_(L) cDNA.

[0016]FIG. 3 is an illustration of the full-length cDNA sequenceencoding human estrogen receptor-β (hERβ_(L)) (SEQ ID NO:1).

[0017]FIG. 4 is an illustration of the predicted amino acid sequence ofthe hERβ polypeptide (SEQ ID NO:2). The first 45 (previously unknown)amino acids are underlined.

[0018]FIG. 5 is a photographic illustration of an autoradiogram of a 10%SDS-polyacrylamide gel in which hERβ in vitro translation products areresolved. Lane 1, hERβ_(T); lane 2, hERβ_(L) produced from a vectorencoding a synthetic translation initiation site; lane 3, hERβ_(L)produced from a vector encoding the natural hERβ translation initiationsequences.

[0019]FIG. 6A is a graphic illustration of the transcriptionalactivation capacity of full-length hERβ (hERβ_(L)) and truncated hERβ(hERβ_(T)) expressed in HepG2 cells. Cells were transfected with eitherhERβ_(L) or hERβ_(T) and co-transfected with a luciferase reporterplasmid containing an estrogen response element (ERE) (ERE.TK.LUC) and acontrol β-galactosidase plasmid. Cells were incubated in the absence orpresence of estradiol, after which luciferase activity was measured andnormalized to β-galactosidase activity. FIG. 6B is a graphicillustration of luciferase activity in HepG2 cells transfected witheither hERβ_(L) or hERβ_(T) and co-transfected with a luciferasereporter plasmid lacking an ERE (TK. LUC)

[0020]FIG. 7A is a graphic illustration of the effect of estradiolstimulation of full-length hERβ (hERβ_(L)) and truncated hERβ (hERβ_(T)) on NFKB activation in HepG2 cells. Cells were transfected with eitherhERβ_(L) or hERβ_(T) and co-transfected with a luciferase reporterplasmid containing three copies of an NFKB binding site (3×-NFkB TK.LUC)and a control β-galactosidase plasmid. Cells were stimulated withinterleukin-1β and incubated in the absence or presence of estradiol,after which luciferase activity was measured and normalized toβ-galactosidase activity. FIG. 7B is a graphic illustration ofluciferase activity in cells transfected with either hERβ_(L) orhERβ_(T) and co-transfected with a luciferase reporter plasmid lackingan NFkB binding site (TK.LUC).

[0021]FIG. 8 is a graphic illustration of the transcriptional activationcapacity of full-length hERβ (hERβ_(L)) and truncated hERβ (hERβ_(T))expressed in HAECT-1 human endothelial cells. Cells were transfectedwith either hERβ_(L) or hERβ_(T) and co-transfected with a luciferasereporter plasmid containing an ERE (ERE.TK.LUC) or one lacking an ERE(TK.LUC). Cells were incubated in the absence or presence of estradiol,after which luciferase activity was measured. ERE TK.LUC values werenorrnalized to TK.LUC values and are presented as mean±S.E. (n=4).

[0022]FIG. 9 is a graphic illustration of the effect of increasing dosesof estrogens (17-β estradiol or genistein) on the transcriptionalactivation capacity of full-length hERβ (hERβ_(L)) and truncated hERβ(hERβ_(T)) expressed in S. cerevisiae. Cells were transformed witheither hERβ_(L) or hERβ_(T) and co-transformed with a β-galactosidasereporter plasmid containing an ERE. Transformed cells were treated withestrogens for 3h and assayed for β-galactosidase activity.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Human estrogen receptor-β (hereinafter, hERβ) comprisesaminoterminal amino acid residues not previously known. The presentinvention encompasses isolated, purified, nucleic acids encodingauthentic full-length hERβ of 530 amino acid residues and fragmentsthereof which include nucleic acids encoding amino acids 1-45 of hERβ.The invention also encompasses isolated, purified, polypeptidescomprising hERβ and peptides derived therefrom, particularly peptideswhich include residues 1-45 of hERβ. The invention also providesexpression systems in which transcriptionally active hERβ or fragmentsderived therefrom can be produced, as well as screening methods foridentifying hERβ agonists and antagonists (including tissue-specificestrogens and anti-estrogens) as well as hERβ co-activators andinhibitors.

[0024] Isolation and Charactenzation of the Gene Encoding hERβ

[0025] The present inventors have isolated the cDNA encoding hERβ usingthe methods outlined below. Human testis Poly A+RNA (1 μg, Clontech,Palo Alto Calif.) was mixed with 0.5 μg oligo dT primer (GIBCO-BRL,Gaithersburg Md.) in a total volume of 10 μl. The mixture was heated at70° C. for 10 minutes, and, after cooling on ice, was supplemented with500 μM of each deoxynucleoside triphosphate, 1X cDNA synthesis buffer,and 10 mM DTT to a final reaction volume of 20 μl. The mixture wasincubated at 42° C. for 2.5 minutes and then supplemented with 1-2 unitsreverse transcriptase (GIBCO-BRL, Gaithersburg Md.), after which it wasincubated at 45° C. for 30 minutes and 50° C. for 5 minutes. One-tenthof this mixture (approximately 2 μl) containing the cDNA template wasthen used in PCR amplification of hERβ using forward and reverse primersas described below.

[0026] Alignment of the known rat ERβ sequence (Kuiper et al., Proc.Natl. Acad. Sci. USA 93:5925, 1996) with that of a human homologue(Mosselman et al., FEBS Letts. 392:49, 1996) suggested that the humansequence lacked at least the ultimate and penultimate residues at itsaminoterminus, as shown below: Rat: MTFYSPAVMNYS . . . Human:--GYSPAVMNYS . . .

[0027] Based on this information, PCR primers were designed thatsupplement the human sequence with the two missing aminoterminalresidues M and T and with an artificial Kozak translation initiationsequence. The forward primer, having the sequence5′-GGAAGCTTGTCGACCATCATGACCGGCTATAGCCCTGCTGTGATG- 3′

[0028] and a reverse primer, having the sequence5′-GGATCTAGAGTCGACGCGTCACTGAGACTGAGGGTTCTGG-3′

[0029] were used to amplify hERβ sequences in a reaction containing thefollowing components: 2 μl of the CDNA template described above; 1X PCRbuffer; 200 μM of each deoxynucleoside triphosphate, 2 units of hot tabpolymerase (Amersham, Arlington Heights Ill.), and 1 μg of each of theforward and reverse primers. The reaction mixture was heated to 95° C.for 2 minutes, annealed at 52° C. for 1 minute, and amplified using 36cycles of 72° C. for 1.5 minutes.

[0030] A fragment of approximately 1500 bp in length was produced. Thefragment digested with HindIII and XbaI (which cleave at sites presentin the forward and reverse primer sequences, respectively, but not inthe main body of the amplified cDNA sequence) and cloned into thecorresponding sites of the pcDNA3 expression vector (Invitrogen,Carlsbad Calif.). This assymetric cloning strategy places the 5′ end ofhERβ cDNA under the control of the viral CMV promoter in pcDNA3 (FIGS. 1and 2). Several insert-containing pcDNA3 clones were identified. PlasmidDNA was prepared from three clones using a plasmid purification kit(Qiagen, Santa Clarita Calif.) and their insert sequences weredetermined by the dideoxy termination method. One clone (designatedR61010-2.24 or Clone 3) was found to contain an insert with a nucleotidesequence identical to the published hERβ sequence (Mosselman et al.,FEBS Letts. 392:49, 1996) and had the following 5′ end structure:       M   T   G  Y . . . CCATC ATG ACC GGC TAT . . .

[0031] This clone was designated “truncated hERβ” or hERβ_(T).

[0032] To verify the aminoterminal and upstream sequence of human hERβ,two independent approaches were taken, as described below.

[0033] (1) 10 μl of a human ovary 5′-Stretch cDNA library (Clontech,Palo Alto Calif.) was mixed with 50 μl of 1X K solution (1X PCR Buffer(GIBCO-BRL, Gaithersburg Md.), 2.5 mM MgCl₂, 0.5% Tween-20, 100 μg/mlProteinase K), and the reaction mixture was incubated at 56° C. for 2hours, then at 99° C. for 10 minutes. 5 μl of this reaction mixture werethen used as template in a nested PCR reation. For the first round, theforward primer (pDR2 sequencing primer, Clontech, Palo Alto Calif.) hadthe sequence 5′-CTGGTAAGTTTAGTCTTTTTGTC-3′, and the reverse primer(hERβ-specific, designated oligo #12908) had the sequence5′-GCTTCACACCAAGGACTCTTTTGAG-3′. The reaction contained 1X Klentaq PCRreaction buffer (40 mM Tricine-KOH, 15 mM KOAc, 3.5 mM Mg(OAc)₂, 75μg/ml bovine serum albumin); 0.2 mM of each dNTP; 0.2 μM of each of theabove primers, and 1 unit of Klentaq Polymerase Mix (Clontech, Palo AltoCalif.). Touchdown PCR conditions were as follows: 5 cycles of 94° C.for 2 seconds and 72° C. for 4 minutes, followed by 30 cycles of 94° C.for 2 seconds and 67° C. for 3 minutes.

[0034] Excess nucleotides and primers were removed from first round PCRreactions by purification over Wizard PCR columns (Promega, MadisonWis.). A second-round PCR reaction was then performed using 2 μl of thepurified first-round reaction mixture. For the second round, the forwardprimer was the pDR2 sequencing primer shown above, and the reverseprimer had the sequence 5′-GTTGGCCACAACACATTTGGGCTTGT-3′ (hERβ-specific,designated oligo #13871). The PCR reaction and cycling conditions wereidentical to those employed in the first round. The products were clonedinto pCR2.1 (Invitrogen) and three resulting clones were sequenced. Allthree clones (designated L1, L2, and L3) contained hERβ inserts ofdifferent lengths, all of which were homologous to hERβ and to eachother.

[0035] (2) A Marathon Ready thymus cDNA kit (Clontech) for 5′ rapidamplification of cDNA ends (RACE) was also used to isolated hERβ 5′clones. In the first round of a nested PCR reaction, 5 μl of humanthymus Marathon-ready cDNA (Clontech) was used as template. The forwardprimer had the sequence 5′-CCATCCTAATACGACTCACTATAGGGC-3′ (Adaptorprimer 1, Clontech), and the reverse primer had the sequence5′-GCTTCACACCAAGGACTCTTTTGAG-3′ (hERβ-specific, designated oligo#12908). The PCR reaction and cycling conditions were identical to thosedescribed in (1) above.

[0036] Excess nucleotides and primers were removed from the first roundPCR reactions by purification over Wizard PCR columns (Promega). Asecond round PCR reaction was performed using 2 μl of the purified firstround reaction. For the second round, the forward primer had thesequence 5′-ACTCACTATAGGGCTCGAGCGGC-3′ (nested adaptor primer 2,Clontech), and the reverse primer had the sequence5′-GTTGGCCACAACACATTTGGGCTTGT-3′ (hERβ-specific, designated oligo#13871). The second round PCR reaction and cycling conditions wereidentical to those employed in the first round. The products were clonedinto the pCR2.1 vector and two clones were sequenced. The two clonescontain insert sequences of different lengths that are homologous tohERβ, to each other, and to the sequences isolated from a human ovarycDNA library as described above.

[0037] All of the hERβ sequences isolated by methods (1) and (2) abovecontained 110 nucleotides corresponding to hERβ_(T) sequences, as wellas 228 additional nucleotides at the 5′ end (FIG. 3).

[0038] The hERβ cDNA sequence determined from these clones containedseveral important differences from the previously known human sequence.First, the third amino acid of the previous sequence was found to be Fand not G (see above). Second, the methionine residue at theaminoterminus of the previous sequence was found not to be the initiator(i.e., true aminoterminal) residue. Rather, the authentic full-lengthhERβ cDNA sequence encodes a polypeptide having 530 residues, the first45 of which are not found in the previously known human sequence (FIG.4). The sequence appears to be quite homologous to rat ERβ; however,this reading frame was not identified previously (Kuiper et al., Proc.Natl. Acad. Sci. USA 93:5925, 1996). Furthermore, an optimal Kozaktranslation initiation sequence is found upstream of the newlydiscovered initiator methionine codon. A termination codon wasidentified 63 nucleotides upstream to the authentic ATG initiator codonin the same reading frame.

[0039] The cDNA encoding authentic full-length hERβ was cloned intopCDNA3 under the control of the CMV promoter; this expression vector wasdesignated “long hERβ” or hERβ_(L).

[0040] Syntliesis of Full-length hERβ and Truncated HERβ

[0041] To examine the natural start site for translation of hERβ, threeplasmids were subjected to coupled transcription-translation, encodinghERβ_(T) (with a synthetic upstream translation initiation sequence),hERβ_(L) (with a synthetic upstream translation initiation sequence),and hERβ_(L) containing 93 nucleotides of its native upstream sequence(the entire sequence shown in FIG. 3). The plasmids were transcribed andtranslated using the TNT T7 Coupled Reticulocyte Lysate System (Promega#L4610). Circular plasmid DNA was purified using Qiagen Maxi-Kit #12362.2 μg of the DNA was transcribed and translated in a single reaction inthe presence of [³⁵S]-methionine (New England Nuclear, Boston Mass.).The translation products were resolved on a 10% SDS polyacrylamide geland were visualized by autoradiography (FIG. 5).

[0042] The resulting translation products of both hERβ_(L) products wereof similar size (˜63 kDa), and the hERβ_(T) product was appropriatelyshorter (˜56 KDa). This indicates that the initiator ATG most likelyutilized in vivo is the ATG at position 94-96. Utilization of a furtherupstream ATG is unlikely because of a termination codon in-fram with thepresumed start site. Confirmation of the authentic start site isachieved by subjecting hERβ polypeptides to aminoterminal sequencing.

[0043] Functional Differences Between Full-length hERβ and TruncatedhERβ

[0044] The experiments described below were performed to evaluate thetranscription activation properties of full-length hERβ according to thepresent invention and to compare it with that of truncated hERβ.hERβ_(L) and hERβ_(T) were expressed in parallel in different cell typesand tested for their ability to transactivate reporter genes containingestrogen response elements (EREs). Alternatively, hERβ_(L) and hERβ_(T)may be expressed in host cells containing endogenous estrogen-responsivegenes and the estrogen-mediated activation of the endogenous genes ismeasured.

[0045] (i) HepG2 Cells:

[0046] HepG2 cells (ATCC) were transfected in parallel with eitherpcDNA3-hERβ_(L) or pcDNA3-hERβ_(T) using the calcium phosphateco-precipitation method. Cells were co-transfected with a reporterplasmid containing a luciferase gene preceded by either an ERE upstreamof the thymidine kinase (TK) basal promoter, or the TK basal promoteralone. Cells also received a plasmid encoding β-galactosidase under thecontrol of an RSV promoter, which was used to correct for variation inDNA uptake. Five hours after transfection, cells were incubated with orwithout 10⁻⁶M 17-β estradiol for 20 hours, after which cell extractswere prepared. Luciferase activity was measured by a chemiluminescentmethod using the Promega luciferase assay system, and β-galactosidaseactivity was measured by Galactolight (Tropix, Inc., Bedford Mass.);luciferase activity was then normalized to β-galactosidase activity.

[0047] The results shown in FIG. 6A indicate that, in the presence ofestradiol, hERβ_(T) caused a 2-fold stimulation of ERE activity. Bycontrast, hERβ_(L) under the same conditions caused a 6-fold stimulationof ERE activity. Thus, hERβ_(L) is about 3-fold more active thanhERβ_(T) in this circumstance.

[0048] In a separate experiment, HepG2 cells weretransfected withhERβ_(L) or hERβ_(T) as above, but the reporter gene consisted of threecopies of an NFkB binding site upstream of the TK basal promoter.Transfected cells were incubated with or without interleukin-1β (IL-1β)to activate NFkB and/or with estradiol prior to luciferasedetermination. The results shown in FIG. 7 indicate that hERβ_(L) wascapable of attenuating the IL-1β-mediated NFkB transcriptionalactivation (to an extent similar to that observed with hERα), whilehERβ_(T) exhibited no inhibitory activity.

[0049] (ii) Human Endothelial Cells:

[0050] HAECT-1 cells (a clonal immortalized human aortic endothelia cellline derived by infection with Ad5 ori-SV40 ts A209) were transfectedwith pcDNA3hERβ_(T) or pcDNA3-hERβ_(L) and ERE-luciferase plasmids byelectroporation. After 4 hours, the cells were treated overnight with orwithout 100 nM 17-β estradiol prior to luciferase activity measurements.The results shown in FIG. 8 indicate that hERβ_(L) is 2-3 times moreactive than hERβ_(T) in activating the ERE-reporter gene in the presenceof estradiol. In independent experiments, cells transfected underidentical conditions were monitored for their levels of estrogenreceptors using a ligand binding assay. The results indicate that theincreased activity of hERβ_(L) relative to hERβ_(T) is not due to anincrease in receptor number or stability, and, further, that the 2-3fold increment measured in the above experiment may be an underestimateof the true transactivational capacity of hERβ.

[0051] (iii) Yeast:

[0052]S. cerevisiae strain BJ2168 (Yeast Genetic Stock Center, BerkeleyCalif.) was co-transformed with an ERE-LacZ reporter plasmid (designatedYRpE2) and yeast vectors expressing either hERβ_(L) or hERβ_(T) underthe control of the yeast triose phosphate isomerase promoter in theyeast pYX242 vector (R&D Systems, Minneapolis Minn.). Transformed cellswere grown in selective medium for 24 hours, after which they weretreated in the presence or absence of increasing concentrations ofeither 17-β estradiol or the phytoestrogen Genistein (ResearchBiochemical International, Natick Mass.) for 3 hours prior todetermination of β-galactosidase activity. The dose-response resultsshown in FIG. 9 indicate that the maximal level of estrogen-stimulatedLacZ expression was 2-fold higher in hERβ_(L)-transformed cells relativeto hERβ_(T)-transformed cells.

[0053] DNA, Vectors, and Expression Systems

[0054] Many conventional techniques in molecular biology, microbiology,and recombinant DNA, are used in practicing the present invention. See,for example, Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

[0055] The present invention encompasses purified, isolated, nucleicacid sequences encoding hERβ, including, e.g., the nucleotide sequencedepicted in FIG. 3 SEQ ID NO:1 and subfragments derived therefrom,including without limitation transcriptional activation-competentfragments. An “isolated” or “purified” nucleic acid is a nucleic acid orpolypeptide that is removed from its original environment (for example,its natural environment if it is naturally occurring). An isolatednucleic acid or polypeptide contains less than about 50%, preferablyless than about 75%, and most preferably less than about 90%, of thecellular components with which it was originally associated.

[0056] A nucleic acid that is “derived from” an hERβ sequence is anucleic acid sequence that corresponds to a region of the sequence,sequences that are homologous or complementary to the sequence, and“sequence-conservative variants” and “function-conservative variants”.Sequence-conservative variants are those in which a change of one ormore nucleotides in a given codon position results in no alteration inthe amino acid encoded at that position. Function-conservative variantsare those in which the amino acid sequence of hERβ has been changedwithout altering the overall conformation and transcriptional activationfunction of the hERβ polypeptide, including, but not limited to,replacement of an amino acid with one having similar physico-chemicalproperties (such as, for example, acidic, basic, hydrophobic, and thelike). A large number of candidate function-conservative hERβ variants,as well as fragments of hERβ that retain transcriptional activationactivity, can be prepared using routine recombinant DNA manipulations aswell as random or site-directed mutagenesis techniques. Furthermore,hERβ-derived variants or fragments that exhibit transcriptionalactivation activity can be identified using routine experimentation byemploying the methods described herein, e.g., by co-expression with anappropriate reporter gene followed by measurement of reporter genetranscription in the presence and absence of an estrogen.

[0057] In another embodiment, the present invention encompassesisolated, purified, nucleic acids comprising nucleotides 94-229 of thesequence depicted in FIG. 3, SEQ ID NO:1, which encode amino acids 1-45of hERβ, and sequence-conservative variants thereof.

[0058] The nucleic acids of the present invention include purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

[0059] The nucleic acids may be isolated directly from cells.Alternatively, PCR can be used to produce the nucleic acids of theinvention, using either chemically synthesized strands or genomicmaterial as templates. Primers used for PCR can be synthesized using thesequence information provided herein and can further be designed tointroduce appropriate new restriction sites, if desirable, to facilitateincorporation into a given vector for recombinant expression.

[0060] The nucleic acids of the present invention may be flanked bynatural regulatory sequences, or may be associated with heterologoussequences, including promoters, enhancers, response elements, signalsequences, polyadenylation sequences, introns, 5′- and 3′- noncodingregions, and the like. The nucleic acids may also be modified by manymeans known in the art. Non-limiting examples of such modificationsinclude methylation, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.). Nucleic acids may contain one or moreadditional covalently linked moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine,etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g.,metals, radioactive metals, iron, oxidative metals, etc.), andalkylators. PNAs are also included. The nucleic acid may be derivatizedby formation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the nucleic acid sequences of thepresent invention may also be modified with a label capable of providinga detectable signal, either directly or indirectly. Exemplary labelsinclude radioisotopes, fluorescent molecules, biotin, and the like.

[0061] The invention also provides nucleic acid vectors comprisinghERβ-encoding sequences or derivatives or fragments thereof. A largenumber of vectors, including plasmid and fungal vectors, have beendescribed for replication and/or expression in a variety of eukaryoticand prokaryotic hosts, and may be used for gene therapy as well as forsimple cloning or protein expression. The encoded hERβ-derivedpolypeptides may be expressed by using many known vectors, such as pUCplasmids, pET plasmids (Novagen, Inc., Madison, Wis.), or pRSET or pREP(Invitrogen, San Diego, Calif.), and many appropriate host cells, usingmethods disclosed or cited herein or otherwise known to those skilled inthe relevant art. The particular choice of vector/host is not criticalto the practice of the invention.

[0062] Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes. The inserted hERβ-encoding sequences may besynthesized by standard methods, isolated from natural sources, orprepared as hybrids, etc. Ligation of the hERβ-encoding sequences totranscriptional regulatory elements and/or to other amino acid codingsequences may be achieved by known methods. Suitable host cells may betransformed/transfected/infected as appropriate by any suitable methodincluding electroporation, CaCl₂ mediated DNA uptake, viralvector-mediated DNA delivery, fungal infection, microinjection,microprojectile, or other established methods.

[0063] Appropriate host cells included bacteria, archebacteria, fungi,especially yeast, and plant and animal cells, especially mammaliancells. Of particular interest are E. coli, B. Subtilis, Saccharomycescerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9cells, C129 cells, 293 cells, Neurospora, and HepG2 cells, CHO cells,COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoidcell lines. Preferred replication systems include M13, Co1E1, SV40,baculovirus, lambda, adenovirus, and the like. A large number oftranscription initiation and termination regulatory regions have beenisolated and shown to be effective in the transcription and translationof heterologous proteins in the various hosts. Examples of theseregions, methods of isolation, manner of manipulation, etc. are known inthe art. Under appropriate expression conditions, host cells can be usedas a source of recombinantly produced hERβ-derived peptides andpolypeptides.

[0064] Advantageously, vectors may also include a transcriptionregulatory element (i.e., a promoter) operably linked to the hERβportion. The promoter may optionally contain operator portions and/orribosome binding sites. Non-limiting examples of bacterial promoterscompatible with E. coli include: β-lactamase (penicillinase) promoter;lactose promoter; tryptophan (trp) promoter; arabinose BAD operonpromoter; lambda-derived P₁ promoter and N gene ribosome binding site;and the hybrid tac promoter derived from sequences of the trp and lacUV5 promoters. Non-limiting examples of yeast promoters include triosephosphate isomerase promoter, 3-phosphoglycerate kinase, promoter,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase(GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase(ADH) promoter. Suitable promoters for mammalian cells include withoutlimitation viral promoters such as that from Simian Virus 40 (SV40),Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus(BPV). Mammalian cells may also require terminator sequences and poly Aaddition sequences and enhancer sequences which increase expression mayalso be included; sequences which cause amplification of the gene mayalso be desirable. Furthermore, sequences that facilitate secretion ofthe recombinant product from cells, including, but not limited to,bacteria, yeast, and animal cells, such as secretory signal sequencesand/or prohormone pro region sequences, may also be included. Thesesequences are well described in the art.

[0065] Nucleic acids encoding hERβ-derived polypeptides may also beintroduced into cells by recombination events. For example, such asequence can be introduced into a cell, and thereby effect homologousrecombination at the site of an endogenous gene or a sequence withsubstantial identity to the gene. Other, recombination-based methodssuch as nonhomologous recombinations or deletion of endogenous genes byhomologous recombination may also be used.

[0066] The nucleic acids of the present invention find use as templatesfor the recombinant production of hERβ-derived peptides or polypeptides.

[0067] hERβ-derived polypeptides

[0068] The present invention encompasses purified hERβ-derivedpolypeptides comprising amino acids 1-45 of hERβ and further comprisingall or part of the amino acid sequence depicted in FIG. 4, SEQ ID NO:2,and function-conservative variants thereof, i.e., variants that exhibitestrogen-induced transcriptional activation activity. Also encompassedby the invention are peptides comprising amino acids 1-45 of SEQ ID NO:2and function-conservative variants thereof.

[0069] Nucleic acids comprising hERβ-coding sequences can be used todirect the expression of hERβ-derived polypeptides in intact cells or incell-free translation systems. The known genetic code, tailored ifdesired for more efficient expression in a given host organism, can beused to synthesize oligonucleotides encoding the desired amino acidsequences. The phosphoramidite solid support method of Matteucci et al.,1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J.Biol. Chem. 764:17078, or other well known methods can be used for suchsynthesis. The resulting oligonucleotides can be inserted into anappropriate vector and expressed in a compatible host organism.

[0070] The polypeptides of the present invention, includingfunction-conservative variants of the disclosed hERβ sequences, may beisolated from wild-type or mutant human cells, or from heterologousorganisms or cells (including, but not limited to, bacteria, fungi,insect, plant, and mammalian cells) into which an hERβ-derivedprotein-coding sequence has been introduced and expressed. Furthermore,the polypeptides may be part of recombinant fusion proteins.

[0071] Polypeptides may be chemically synthesized by commerciallyavailable automated procedures, including, without limitation, exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. The polypeptides arepreferably prepared by solid phase peptide synthesis as described byMerrifield, 1963, J. Am. Chem. Soc. 85:2149.

[0072] “Isolation” or “purification” of an hERβ-derived polypeptiderefers to the isolation of the polypeptide in a form that allows itstranscriptional activation activity to be measured without interferenceby other components of the cell in which the polypeptide is expressed.Methods for polypeptide purification are well-known in the art,including, without limitation, preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ionexchange and partition chromatography, and countercurrent distribution.For some purposes, it is preferable to produce the polypeptide in arecombinant system in which the hERβ-derived protein contains anadditional sequence tag that facilitates purification, such as, but notlimited to, a polyhistidine sequence. The polypeptide can then bepurified from a crude lysate of the host cell by chromatography on anappropriate solid-phase matrix. Alternatively, antibodies producedagainst an hERβ-derived protein or against peptides derived therefromcan be used as purification reagents. Other purification methods arepossible.

[0073] The present invention also encompasses derivatives and homologuesof hERβ-encoded polypeptides. For some purposes, nucleic acid sequencesencoding the peptides may be altered by substitutions, additions, ordeletions that provide for functionally equivalent molecules, i.e.,function-conservative variants. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofsimilar properties, such as, for example, positively charged amino acids(arginine, lysine, and histidine); negatively charged amino acids(aspartate and glutamate); polar neutral amino acids; and non-polaramino acids.

[0074] The isolated polypeptides may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.They may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

[0075] hERβ-specific Antibodies

[0076] The present invention encompasses antibodies that specificallyrecognize hERβ-derived peptides and polypeptides, including withoutlimitation antibodies that recognize hERβ but not, e.g., hERα, and thosethat recognize hERβ_(L) but not hERβ_(T). Such hERβ-specific antibodiescan be used conventionally, e.g., as diagnostic reagents or as reagentsfor purification of hERβ-derived polypeptides. Other uses includeimmunocytochemical localization of hERβ; gel shift assays; and“pull-down” experiments to identify protein co-activators associatedwith hERβ.

[0077] hERβ-specific antibodies according to the present inventioninclude polyclonal and monoclonal antibodies. The antibodies may beelicited in an animal host by immunization with hERβ immunogeniccomponents or may be formed by in vitro immunization (sensitization) ofimmune cells. The immunogenic components used to elicit the productionof antibodies may be isolated from any cell source or may be chemicallysynthesized. The antibodies may also be produced in recombinant systemsprogrammed with appropriate antibody-encoding DNA. Alternatively, theantibodies may be constructed by biochemical reconstitution of purifiedheavy and light chains. The antibodies include hybrid antibodies (i.e.,containing two sets of heavy chain/light chain combinations, each ofwhich recognizes a different antigen), chimeric antibodies (i.e., inwhich either the heavy chains, light chains, or both, are fusionproteins), and univalent antibodies (i.e., comprised of a heavychain/light chain complex bound to the constant region of a second heavychain). Also included are Fab fragments, including Fab′ and F(ab)₂fragments of antibodies. Methods for the production of all of the abovetypes of antibodies and derivatives are well-known in the art. Forexample, techniques for producing and processing polyclonal antisera aredisclosed in Mayer and Walker, 1987, Immunochemical Methods in Cell andMolecular Biology, (Academic Press, London). The general methodology formaking monoclonal antibodies by hybridomas is well known. Immortalantibody-producing cell lines can be created by cell fusion, and also byother techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. See, e.g.,Schreier et al., 1980, Hybridoma Techniques. Panels of monoclonalantibodies produced against hERβ epitopes can be screened for variousproperties; i.e., for isotype, epitope affinity, etc.

[0078] Antibodies against hERβ-derived immunogenic components can beused, unlabeled or labeled by standard methods, as the basis forimmunoassays. The particular label used will depend upon the type ofimmunoassay used. Examples of labels that can be used include but arenot limited to radiolabels such as ³²P, ¹²⁵I, ³H and ¹⁴C; fluorescentlabels such as fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and umbelliferone; chemiluminescers such asluciferia and 2,3-dihydrophthal-azinediones; and enzymes such ashorseradish peroxidase, alkaline phosphatase, lysozyme andglucose-6-phosphate dehydrogenase.

[0079] Applications

[0080] The methods and compositions of the present invention can be usedto identify compounds that interact with hERβ, either to activate or toinhibit its transcriptional activation function. Such compounds include,without limitation, co-activator proteins, as well as estrogens andother steroids, steroid-like molecules, or non-steroid-like moleculesthat act as agonists or antagonists. Screening methods can also be usedto identify tissue-specific estrogens.

[0081] Identification of hERβ-interactive compounds can be achieved bycell-free or cell-based assays. In one set of embodiments, purified hERβis contacted with a labelled ligand, such as, e.g., 17-β estradiol, inthe presence of test compounds to form test reactions, and in theabsence of test compounds to form control reactions. The labelled moietymay comprise a radiolabel (such as, e.g., ³H or ¹²⁵I) or a fluorescentmolecule. Incubation is allowed to proceed for a sufficient time andunder appropriate conditions to achieve specific binding, after whichbinding of labelled estradiol to hERβ is measured (by monitoring, e.g.,radioactivity, flurorescence, or fluorescence polarization). In oneembodiment, hERβ produced in E. coli (as described in Example 1 below)is adsorbed to the wells of a microtiter dish and incubated with[³H]-17β estradiol in the absence or presence of test compounds (see,e.g., Example 2 below). Alternatively, soluble receptor is incubatedwith the labelled ligand in the absence or presence of test compounds,and bound ligand is separated from free ligand, either by filtration onglass fiber filters or by using dextran-coated charcoal. See, e.g.,Hulme, ed., Receptor-ligand Interactions: A Practical Approach, IRLPress, NY, 1992).

[0082] Whole cell binding assays may also be used in which bound ligandis separated from free ligand by rinsing. Cells used in these assays mayeither contain endogenous receptor, or may overexpress the receptorsubsequent to stable or transient transfection or infection of an hERβgene or cDNA. Non-limiting examples of suitable cells include COS cells,Hela cells, CHO cells, human umbilical vein endothelial cells (HUVEC),and yeast. Once a compound has been identified as an hERβ-interactivecompound by its binding activity, further in vivo and in vitro tests maybe performed to determine the nature and extent of activity, i.e., as anagonist or antagonist (see below).

[0083] hERβ-interactive compounds may also be identified usingcell-based assays that measure transcriptional activation or suppressionof endogenous or transfected estrogen-responsive genes. For example,agonists (such as, e.g., 17β-estradiol) block interleukin-1β inductionof endogenous E-selectin in primary human umbilical vein endothelialcells (HUVEC) that express hERβ. Antagonists (such as, e.g., ICI-182780)block the agonist activity of 17β-estradiol. Non-limiting examples ofother suitable endogenous estrogen-responsive promoter elements includethose that regulate endothelin-1 (ET-1); HDL receptor (scavengerreceptor type II); and enzymes involved in coagulation and fibrinolysis(such as, e.g., plasminogen activator inhibitor-1 and complement C3).Any promoter element that responds to estrogen may be used as anappropriate target, including, e.g., the NFkB binding site or theapolipoprotein A1 gene enhancer sequence.

[0084] In one set of embodiments, appropriate host cells are transfectedwith an expression vector encoding hERβ and the transfectants areincubated with or without estradiol in the presence or absence of testcompounds. hERβ activity is assessed by measuring transcriptionalactivation of the target sequence. This may be achieved by detection ofmRNA (using, e.g., Northern blot analysis) and/or by detection of theprotein (using, e.g., immunoassays or functional assays). If activationof the target sequence initiates a biochemical cascade, downstreambiological events may also be measured to quantify hERβ activity.hERβ-interactive compounds are identified as those that positively ornegatively influence target sequence activation.

[0085] In another set of embodiments, appropriate host cells(preferably, bacterial or yeast cells) are co-transfected with anexpression vector encoding hERβ and a reporter plasmid containing areporter gene downstream of one or more estrogen response elements(EREs). Transfected cells are incubated with or without estradiol in thepresence of absence of test compounds, after which hERβ activity isdetermined by measuring expression of the reporter gene. In a preferredembodiment, hERβ activity is monitored visually. Non-limiting examplesof suitable reporter genes include luciferase, chloramphenicol acetyltransferase (CAT), and green fluorescence protein.

[0086] Preferably, the methods of the present invention are adapted to ahigh-throughput screen, allowing a multiplicity of compounds to betested in a single assay. Candidate estrogens and estrogen-likecompounds include without limitation diethylstilbesterol, genistein, andestrone. Other hERβ-interactive compounds may be found in, for example,natural product libraries, fermentation libraries (encompassing plantsand microorganisms), combinatorial libraries, compound files, andsynthetic compound libraries. For example, synthetic compound librariesare commercially available from Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich Chemical Company, Inc. (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from, forexample, Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or arereadily producible. Additionally, natural and synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical, and biochemical means (Blondelle et al., TibTech14:60, 1996 ). hERβ binding assays according to the present inventionare advantageous in accommordating many different types of solvents andthus allowing the testing of compounds from many sources.

[0087] Compounds identified as hERβ agonists or antagonists using themethods of the present invention may be modified to enhance potency,efficacy, uptake, stability, and suitability for use in therapeuticapplications, etc. These modifications are achieved and tested usingmethods well-known in the art.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] The following examples are intended to illustrate the presentinvention without limitation.

EXAMPLE 1

[0089] High-level Expression of Human hERβ in E. coli

[0090] Human hERβ according to the present invention is overexpressed inE. coli strain BL21(DE3) using, for example, the pET15B vector. A 10-mlovernight culture is used to inoculate 1 liter LB medium containing 100μg/ml ampicillin. Cultures are grown at 37° C. and then induced by theaddition of 1 mM IPTG. After an additional incubation for 2h at 25° C.,cells are harvested by centrifugation at 10,000×G for 30 minutes andresuspended in 100 ml of a buffer containing 50 mM Tris-HCl, pH 7.4-150mM NaCl. Cells are lysed using a French press, and insoluble material ispelleted by centrifugation. The supernatant solution is recovered andstored at −70° C.

EXAMPLE 2

[0091] Estrogen Receptβ Ligand Binding Assay

[0092] For determining the ability of a particular compound to bindhERβ, 100 μl of the receptor preparation described in Example 1 above,diluted in assay buffer (Dulbecco's phosphate buffered saline (Gibco#14200-075) supplemented with 1 mM EDTA), is added to each well of ahigh-binding masked microtiter plate (Wallac #1450-511, GaithersburgMd.). 10 μl of test compound (or vehicle) and 10 μl of[³H]-17β-estradiol are added to each well, and the plate is incubated atroom temperature for 4-6 hours. Unbound material is aspirated, and theplate is washed three times with 300 μl of assay buffer. Then, 150 μl ofscintillation cocktail (Optiphase Supermix, Wallac #1200-439) is addedper well, and the plate is sealed and agitated for at least 5 min. Boundradioactivity is measured by scintillation counting.

[0093] Test compounds are initially tested at a concentration of 1.5μg/ml (approximately 5 μM for a compound having a molecular mass of300). Positive compounds are then re-tested at a number of differentconcentrations to determine the IC₅₀.

[0094] Data are expressed as percent inhibition of specific binding.Exploratory data analysis (EDA) is performed on raw data to check fornon-normality and non-homogeneity of variance. The maximum likelihoodBox-Cox transformation, which maximizes the normality, homogeneity ofvariance, and goodness of fit of the data, is then obtained. Based onthe result, the appropriate transformation of the data (notransformation, square root transformation, or logarithmictransformation) is used for model fitting. The Huber M-estimator is usedto down weight any outlying transformed observations for analysis ofvariance and dose-response curve fitting.

[0095] For ANOVA, multiple comparisons LSD p-values are computed.Re-transformed summary statistics (mean, s.d, s.e.m.) are obtained foreach treatment group.

[0096] For dose-response curve fitting, a four parameter logistic modelon the transformed, weighted data are fit. The four parameters are min,max, slope, and ED50, where ED50 is defined as the dose whichcorresponds to midway between the estimated max and min. All of theparameters and confidence intervals are re-transformed back to theoriginal units of the data. A further transformation into percentinhibition (using estimated min and max) is performed.

[0097] Using this assay, the following values were obtained forreference compounds: IC₅₀ 95% confidence limits 17β-estradiol 6.7 nM  6-7.5 nM diethylstilbestrol  21 nM  14-31 nM genistein 1.6 nM 1.4-1.8nM

[0098] All patents, applications, articles, publications, and testmethods mentioned above are hereby incorporated by reference.

[0099] Many variations of the present invention will suggest themselvesto those skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

1 13 1 1686 DNA Human 1 cagccattat acttgcccac gaatctttga gaacattataatgacctttg tgcctcttct 60 tgcaaggtgt tttctcagct gctatctcaa gacatggatataaaaaactc accatctagc 120 cttaattctc cttcctccta caactgcagt caatccatcttacccctgga gcacggctcc 180 atatacatac cttcctccta tgtagacagc caccatgaatatccagccat gacattctat 240 agccctgctg tgatgaatta cagcattccc agcaatgtcactaacttgga aggtgggcct 300 ggtcggcaga ccacaagccc aaatgtgttg tggccaacacctgggcacct ttctccttta 360 gtggtccatc gccagttatc acatctgtat gcggaacctcaaaagagtcc ctggtgtgaa 420 gcaagatcgc tagaacacac cttacctgta aacagagagacactgaaaag gaaggttagt 480 gggaaccgtt gcgccagccc tgttactggt ccaggttcaaagagggatgc tcacttctgc 540 gctgtctgca gcgattacgc atcgggatat cactatggagtctggtcgtg tgaaggatgt 600 aaggcctttt ttaaaagaag cattcaagga cataatgattatatttgtcc agctacaaat 660 cagtgtacaa tcgataaaaa ccggcgcaag agctgccaggcctgccgact tcggaagtgt 720 tacgaagtgg gaatggtgaa gtgtggctcc cggagagagagatgtgggta ccgccttgtg 780 cggagacaga gaagtgccga cgagcagctg cactgtgccggcaaggccaa gagaagtggc 840 ggccacgcgc cccgagtgcg ggagctgctg ctggacgccctgagccccga gcagctagtg 900 ctcaccctcc tggaggctga gccgccccat gtgctgatcagccgccccag tgcgcccttc 960 accgaggcct ccatgatgat gtccctgacc aagttggccgacaaggagtt ggtacacatg 1020 atcagctggg ccaagaagat tcccggcttt gtggagctcagcctgttcga ccaagtgcgg 1080 ctcttggaga gctgttggat ggaggtgtta atgatggggctgatgtggcg ctcaattgac 1140 caccccggca agctcatctt tgctccagat cttgttctggacagggatga ggggaaatgc 1200 gtagaaggaa ttctggaaat ctttgacatg ctcctggcaactacttcaag gtttcgagag 1260 ttaaaactcc aacacaaaga atatctctgt gtcaaggccatgatcctgct caattccagt 1320 atgtaccctc tggtcacagc gacccaggat gctgacagcagccggaagct ggctcacttg 1380 ctgaacgccg tgaccgatgc tttggtttgg gtgattgccaagagcggcat ctcctcccag 1440 cagcaatcca tgcgcctggc taacctcctg atgctcctgtcccacgtcag gcatgcgagt 1500 aacaagggca tggaacatct gctcaacatg aagtgcaaaaatgtggtccc agtgtatgac 1560 ctgctgctgg agatgctgaa tgcccacgtg cttcgcgggtgcaagtcctc catcacgggg 1620 tccgagtgca gcccggcaga ggacagtaaa agcaaagagggctcccagaa cccacagtct 1680 cagtga 1686 2 530 PRT Human 2 Met Asp Ile LysAsn Ser Pro Ser Ser Leu Asn Ser Pro Ser Ser Tyr 1 5 10 15 Asn Cys SerGln Ser Ile Leu Pro Leu Glu His Gly Ser Ile Tyr Ile 20 25 30 Pro Ser SerTyr Val Asp Ser His His Glu Tyr Pro Ala Met Thr Phe 35 40 45 Tyr Ser ProAla Val Met Asn Tyr Ser Ile Pro Ser Asn Val Thr Asn 50 55 60 Leu Glu GlyGly Pro Gly Arg Gln Thr Thr Ser Pro Asn Val Leu Trp 65 70 75 80 Pro ThrPro Gly His Leu Ser Pro Leu Val Val His Arg Gln Leu Ser 85 90 95 His LeuTyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser 100 105 110 LeuGlu His Thr Leu Pro Val Asn Arg Glu Thr Leu Lys Arg Lys Val 115 120 125Ser Gly Asn Arg Cys Ala Ser Pro Val Thr Gly Pro Gly Ser Lys Arg 130 135140 Asp Ala His Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser Gly Tyr His 145150 155 160 Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys ArgSer 165 170 175 Ile Gln Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn GlnCys Thr 180 185 190 Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys ArgLeu Arg Lys 195 200 205 Cys Tyr Glu Val Gly Met Val Lys Cys Gly Ser ArgArg Glu Arg Cys 210 215 220 Gly Tyr Arg Leu Val Arg Arg Gln Arg Ser AlaAsp Glu Gln Leu His 225 230 235 240 Cys Ala Gly Lys Ala Lys Arg Ser GlyGly His Ala Pro Arg Val Arg 245 250 255 Glu Leu Leu Leu Asp Ala Leu SerPro Glu Gln Leu Val Leu Thr Leu 260 265 270 Leu Glu Ala Glu Pro Pro HisVal Leu Ile Ser Arg Pro Ser Ala Pro 275 280 285 Phe Thr Glu Ala Ser MetMet Met Ser Leu Thr Lys Leu Ala Asp Lys 290 295 300 Glu Leu Val His MetIle Ser Trp Ala Lys Lys Ile Pro Gly Phe Val 305 310 315 320 Glu Leu SerLeu Phe Asp Gln Val Arg Leu Leu Glu Ser Cys Trp Met 325 330 335 Glu ValLeu Met Met Gly Leu Met Trp Arg Ser Ile Asp His Pro Gly 340 345 350 LysLeu Ile Phe Ala Pro Asp Leu Val Leu Asp Arg Asp Glu Gly Lys 355 360 365Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr 370 375380 Ser Arg Phe Arg Glu Leu Lys Leu Gln His Lys Glu Tyr Leu Cys Val 385390 395 400 Lys Ala Met Ile Leu Leu Asn Ser Ser Met Tyr Pro Leu Val ThrAla 405 410 415 Thr Gln Asp Ala Asp Ser Ser Arg Lys Leu Ala His Leu LeuAsn Ala 420 425 430 Val Thr Asp Ala Leu Val Trp Val Ile Ala Lys Ser GlyIle Ser Ser 435 440 445 Gln Gln Gln Ser Met Arg Leu Ala Asn Leu Leu MetLeu Leu Ser His 450 455 460 Val Arg His Ala Ser Asn Lys Gly Met Glu HisLeu Leu Asn Met Lys 465 470 475 480 Cys Lys Asn Val Val Pro Val Tyr AspLeu Leu Leu Glu Met Leu Asn 485 490 495 Ala His Val Leu Arg Gly Cys LysSer Ser Ile Thr Gly Ser Glu Cys 500 505 510 Ser Pro Ala Glu Asp Ser LysSer Lys Glu Gly Ser Gln Asn Pro Gln 515 520 525 Ser Gln 530 3 12 PRT Rat3 Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser 1 5 10 4 10 PRT Human4 Gly Tyr Ser Pro Ala Val Met Asn Tyr Ser 1 5 10 5 45 DNA ArtificialSequence PCR Primer 5 ggaagcttgt cgaccatcat gaccggctat agccctgctg tgatg45 6 40 DNA Artificial Sequence PCR Primer 6 ggatctagag tcgacgcgtcactgagactg agggttctgg 40 7 4 PRT Human 7 Met Thr Gly Tyr 1 8 17 DNAHuman 8 ccatcatgac cggctat 17 9 23 DNA Artificial Sequence PCR Primer 9ctggtaagtt tagtcttttt gtc 23 10 25 DNA Artificial Sequence PCR Primer 10gcttcacacc aaggactctt ttgag 25 11 26 DNA Artificial Sequence PCR Primer11 gttggccaca acacatttgg gcttgt 26 12 27 DNA Artificial Sequence PCRPrimer 12 ccatcctaat acgactcact atagggc 27 13 23 DNA Artificial SequencePCR Primer 13 actcactata gggctcgagc ggc 23

1. (canceled).
 2. (Canceled).
 3. (Canceled).
 4. (Canceled). 5.(Canceled).
 6. (Canceled).
 7. (Canceled).
 8. (Canceled).
 9. (Canceled).10. (Canceled).
 11. (Canceled).
 12. (Canceled).
 13. (Canceled). 14.(Canceled).
 15. (Canceled).
 16. (Canceled).
 17. (Canceled).
 18. Anisolated estrogen recepter-β comprising amino acids 1-45 of the sequencedepicted in FIG. 4 SEQ ID No.
 2. 19. (Canceled).
 20. (Canceled). 21.(Canceled).
 22. (Canceled).
 23. An antibody that specifically recognizeshERβ.
 24. A method for identifying hERβ-interactive compounds, saidmethod comprising: (a) contacting the polypeptide of claim 18, whichpolypeptide encodes hERβ, with a labeled ligand in the presence of testcompounds, to form test reactions, and in the absence of test compounds,to form control reactions; (b) incubating said test and controlreactions under appropriate conditions to achieve equilibrium binding ofsaid labeled ligand to hERβ; (c) determining the level of binding ofsaid labeled ligand to hERβ in said test and control cultures; and (d)identifying as a hERβ-interactive compound any compound that reduces thebinding of said labeled ligand to hERβ.
 25. A method as defined in claim24, wherein said ligand is 17-β estradiol.
 26. A method as defined inclaim 24, wherein said hERβ-interactive compound is an agonist.
 27. Amethod as defined in claim 24, wherein said hERβ-interactive compound isan antagonist.
 28. (Canceled).
 29. (Canceled).
 30. (Canceled).
 31. Thepolypeptide of claim 18, wherein the polypeptide is modified with alabel capable of providing a detectable signal.
 32. The polypeptide ofclaim 31, wherein the label is a radioisotope.
 33. The polypeptide ofclaim 31, wherein the label is a fluorescent compound.
 34. (Canceled).35. (Canceled).
 36. The polypeptide of claim 18, wherein the polypeptideis produced in intact cells.
 37. The polypeptide of claim 18, whereinthe polypeptide is produced in cell-free translation systems. 38.(Canceled).
 39. (Canceled).
 40. The polypeptide of claim 18, wherein thepolypeptide is chemically synthesized.
 41. The polypeptide of claim 18,wherein the polypeptide is produced in a recombinant system.
 42. Apurified polypeptide comprising amino acids 1-45 of the sequencedepicted in FIG. 4 SEQ ID NO:2, wherein when this polypeptide forms theN-terminus of human estrogen receptor β, the estrogen receptor βstimulates estrogen response element (ERE) activity to a greater extentthan the truncated estrogen receptor lacking this N-terminal polypeptidesequence.
 43. A purified polypeptide comprising amino acids 1-45 of thesequence depicted in FIG. 4 SEQ ID NO:2, wherein when this polypeptideforms the N-terminus of human estrogen receptor β, the estrogen receptorβ attenuates NF-kB transcription activation while the truncated estrogenreceptor lacking this N-terminal polypeptide sequence does not.
 44. Apurified polypeptide comprising amino acids 1-45 of the sequencedepicted in FIG. 4 SEQ ID NO:2, wherein when this polypeptide forms theN-terminus of human estrogen receptor β, the estrogen receptor β is 2 to3 times more active than the truncated estrogen receptor lacking thisN-terminal polypeptide sequence in activating the ERE-reporter gene inthe presence of estradiol.
 45. An isolated estrogen receptor-βcomprising an N-terminus having amino acids 1-45 of the sequencedepicted in FIG. 4 SEQ ID NO:2, wherein the estrogen receptor βstimulates ERE activity to a greater extent than the truncated estrogenreceptor lacking this N-terminal polypeptide sequence.
 46. An isolatedestrogen receptor-β comprising an N-terminus having amino acids 1-45 ofthe sequence depicted in FIG. 4 SEQ ID NO:2, wherein the estrogenreceptor β attenuates NF-kB transcription activation while the truncatedestrogen receptor lacking this N-terminal polypeptide sequence does not.47. An isolated estrogen receptor-β comprising an N-terminus havingamino acids 1-45 of the sequence depicted in FIG. 4 SEQ ID NO:2, whereinthe estrogen receptor β is 2 to 3 times more active than the truncatedestrogen receptor lacking this N-terminal polypeptide sequence inactivating the ERE-reporter gene in the presence of estradiol.